Zinc transporters regulate subcellular zinc distributions with sharp transmembrane zinc gradients among intracellular organelles. The spatiotemporal zinc dynamics provides crucial cellular signaling opportunities, but also challenges intracellular zinc homeostasis with broad disease implications. Our long-term goal is to understand structures and mechanisms of zinc transporters and their functional regulations. This proposal is a competitive renewal of a continuing research project focusing on zinc-efflux transporters from the Cation Diffusion Facilitator (CDF) family. In the past decade, we have elucidated the molecular architecture, coordination chemistry and structural dynamics of a bacterial CDF homolog. While we are making progress on the basic research front, a human CDF, hZnT8 has been identified as a major autoantigen in type-1 diabetes as well as a major risk factor associated with type-2 diabetes. A critical barrier to therapeutic translation of hZnT8 is the lack of knowledge about the molecular mechanism responsible for diabetes association and the functional consequences of susceptibility hZnT8 variations. To fill in this knowledge gap, our research focus in the next budget cycle will shift from a bacterial CDF model to hZnT8 with four specific aims: (1) to understand the mechanism of allosteric regulation of zinc transport. (2) to characterize transport kinetics of purified hZnT8 and its genetic variants, (3) to define the functional roles of hZnT8 and its genetic variants in pancreatic beta cells, (4) to develop hZnT8-specifc mAbs and determine their effects on insulin storage and secretion. The planned research will revolve around functional regulation of CDFs to understand how allosteric regulation of zinc transport occurs in real time (aim-1), how hZnT8 is affected by diabetes-associated genetic variations (aim-2), how hZnT8 modulates vesicular zinc dynamics and insulin secretion in response to glucose stimulations (aim-3), and whether hZnT8 can be blocked by conformation-specific monoclonal antibodies targeting extracellular epitopes (aim-4). The knowledge gained will be used to elucidate the molecular underpinning of hZnT8-based diabetes therapy.